Immunotherapy of patients with metastatic malignant melanoma using tumor infiltrating lymphocytes (TIL) and interleukin-2 has resulted in significant clinical regressions. Our studies are focused on lymphocytes infiltrating breast carcinomas. The goal of this work is to identify breast cancer specific TIL and to characterize the tumor antigens they recognize. We expect to translate these findings into therapies for patients with this disease. We have cultured and studied TIL from 54 breast cancer patients (primary or metastatic sites). No culture has shown specific tumor cytolysis. However, three CD4+ cultures have shown specific cytokine secretion when stimulated with fresh autologous tumor, suggesting that they recognize unique tumor antigens. 1. Screening of breast cancer infiltrating lymphocytes. TIL have been cultured in limiting dilution conditions with and without tumor stimulation, anti-CD3 antibody, PMA and calcium ionophore; CD8+ cytolytic lympohocytes (CTL) with specific tumor reactivity have not been generated. We are studying culture conditions and means of in-vitro T cell activation that will result in specific CTL generation (which at present is common only in melanoma). 2. Establishment of tumor lines. The long-term culture of fresh breast cancer cells derived from patients has been largely unsuccessful in ours and many other laboratories. The short supply of tumor cells is a limitation to our studies. Hence, our recent approach has been to immortalize short-term tumor cultures with the retroviral vector CRIP E6E7. In 4 of 9 tumors cultured and transduced, a long-term line has been established. In parallel we have developed fibroblast and B cell lines, all of which are necessary for lymphocyte cloning and testing. In summary, we are developing and studying biologic reagents that we anticipate will enhance our understating of immune mediated cancer regression.